Effects of the estrous cycle stage on the prolactin secretory response to dopamine in vitro

Dopamine (DA) will both stimulate and inhibit prolactin (PRL) secretion from the anterior pituitary gland in vitro and in vivo. The present study was designed to determine if there are selected times during the estrous cycle of the rat when one function is favored over the other. Anterior pituitary glands collected on diestrus-1 (D1), diestrus-2 (D2), the morning of proestrus (Pro-AM), the afternoon of proestrus (Pro-PM), and estrus (E) were enzymatically dissociated and placed in monolayer culture. On the fourth day in culture, cells were challenged for 10, 20, 30, 60, 120, 180, or 240 min with media alone or media containing either 100 pM or 1 μM DA. The concentration of PRL in the media was determined by radioimmunoassay. Regression analysis revealed that in the absence of DA, PRL secretion from cultured cells differed significantly depending on the stage of the estrous cycle during which they were obtained. Cells obtained during the morning of diestrus-2 secreted PRL at the greatest rate compared to other stages of the cycle. When all stages were compared, the rates of PRL secretion were: D2>E>D1>Pro-AM>Pro-PM (each significantly different from the others,P<0.01). By 20–30 min of exposure to 100 pM DA, the rate of PRL secretion from cells obtained during each stage of the cycle was significantly enhanced. This enhanced secretion persisted in cells obtained during D2 and Pro-PM but was short-lived in cells obtained during other stages. No inhibition of PRL secretion was induced by this dose of DA. PRL secretion was inhibited when treated with 1 μM DA in cells obtained at all stages of the estrous cycle. Inhibition was more prolonged in cells obtained on D1, D2, and Pro-AM. DA was least effective as an inhibitor of PRL secretion in cells obtained during Pro-PM and E. Prior to inhibiting PRL secretion in cells obtained during Pro-PM, 1 μM DA rapidly stimulated PRL secretion. This effect persisted for 60 min. These data suggest that in the absence of DA, the dynamics of PRL secretion from anterior pituitary cells in vitro differ depending on the stage of the estrous cycle during which the cells were obtained. Moreover, the in vivo environment of the cell determines the direction and magnitude of the PRL-secretory response to DA.     

Changes of pituitary thyrotropin releasing hormone (TRH) receptor level and prolactin response to TRH during the rat estrous cycle.

The plasma PRL and TSH responses to TRH injected iv at different stages of the estrous cycle in normal rats under Surital anesthesia were maximal during the afternoon of proestrus and morning of estrus and lowest on diestrus I. As calculated from the areas under the plasma response curves, a 10-fold difference was found between the maximal and minimal PRL responses while a 2-fold difference was measured for TSH. The plasma PRL and TSH responses to TRH showed a correlation with the binding of [3H]TRH to anterior pituitary gland, a 3-fold difference being observed between the minimal binding measured on the morning of diestrus II and the maximal value found on the evening of proestrus. Contrary to findings with LHRH and LH, repeated injections of a small dose (10 ng) of TRH in the afternoon of proestrus abolished PRL and TSH responses to subsequent injection of the neurohormone.     

Bioactivity of serum and pituitary prolactin during the rat estrous cycle

The availability of a mitogenic bioassay for PRL in which multiple, small samples could be analyzed led us to examine circulating and pituitary PRL bioactivity during the estrous cycle of the rat. Bio- (B) and immuno (I)-activities were compared in individual samples using rat PRL RP-1 standard. B:I ratios ranged between 2.3 and 6.8 for serum and 1.4 and 3.2 for pituitaries. Serum bioactivity increased 24-fold during the surge of PRL on the afternoon of proestrus, whereas pituitary bioactivity decreased almost 5-fold during this time period. Serum B:I ratios were significantly (P less than 0.01) elevated on the day of proestrus (0900-1800 h) compared to those on diestrous day 2. Pituitary B:I ratios ranged from a high of 2.3 +/- 0.3 on diestrous day 2 and 1500 h on proestrus to 1.8 +/- 0.1 on diestrous day 1. Differences between mean B:I ratios of pituitaries obtained at different cycle stages were not statistically significant. Interestingly, serum B and I were not correlated (r = 0.55, 0.68) at the peak of the proestrous PRL surge when pituitary extracts exhibited the highest correlation (r = 0.94). The augmented bioactivity of serum compared to that in pituitaries was not due to a synergistic serum effect. It is likely that the proestrous PRL surge, similar to the preovulatory LH surge, results from secretion of a pool of high biological activity. Since no evidence was found for a lactogen synergist in the circulation, the difference between serum and pituitary bioactivity could reflect an averaging of releasable and nonreleasable pools of PRL with varying degrees of biopotency. That these differences result from postsecretion enhancement of PRL biopotency, possibly due to changes in molecular structure and/or conformation, also cannot be ruled out. PRL biopotency in the circulation of the rat is much higher than that reported for women, perhaps reflecting the physiological relevance of PRL to luteal function in the rat.     

Changes of pituitary sensitivity to LH-RH during the rat estrous cycle

The plasma LH (luteinizing hormone) response to 200 ng of LH-RH (LH-releasing hormone) injected subcutaneously at different stages of the estrous cycle in normal rats under Surital anesthesia was maximal during the afternoon of proestrus and lowest on diestrus I. The area under the plasma LH curve measured at 13:00 on proestrus was approximately 7-fold higher than that obtained at 15:30 h on diestrus I. Intermediate responses were found on diestrus II, estrus and morning of proestrus. An approximately 2.5-fold higher LH response was observed on proestrus than on diestrus I after injection of [D-Ala²], des Gly-NH₂¹⁰] LH-RH ethylamide at 15:00 h. That these marked changes of LH response are not secondary to interference by endogenous LH-RH, changes of the metabolism or transport of exogenous LH-RH or modification of plasma LH clearance is ascertained by the finding of similar changes of pituitary sensitivity to LH-RH under in vitro conditions using pituitaries collected at the same stages of the estrous cycle. As measured both in vivo and in vitro, not only the amplitude but also the speed of LH response are maximal during the afternoon of proestrus and minimal on diestrus I.     

Changes in tuberoinfundibular dopaminergic neuron activity during the rat estrous cycle in relation to the prolactin surge: alteration by a mammary carcinogen

An attempt was made to correlate the physiological or the dimethylbenz(a)anthracene (DMBA)-enhanced serum prolactin (PRL) surge, which occurs in the afternoon of proestrus in female Sprague-Dawley (SD) rats, with physiological or pathological changes in two biochemical estimates of the tuberoinfundibular dopaminergic (TIDA) neuron activity. Dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) concentrations as well as tyrosine hydroxylase (TH) activity were measured in the median eminence (ME) of control or DMBA-pretreated SD rats throughout the estrous cycle in relation to PRL secretion. In both groups of females, while the DA content was fairly constant, the DOPAC content and TH activity in the ME fluctuated markedly throughout the estrous cycle. Thus, in control animals, the DOPAC content, DOPAC/DA ratio and TH activity which were stable on the days of diestrus and morning of proestrus were markedly decreased at noon and early afternoon when serum PRL levels began to rise. Later in the afternoon of proestrus, when serum PRL levels were maximal, there was a marked but transient increase in the DOPAC content and DOPAC/DA ratio as well as a brief surge in TH activity. In the evening of the same day, when serum PRL returned to basal levels, the DOPAC content, DOPAC/DA ratio and TH activity were low. Finally on estrus morning, the DOPAC content, DOPAC/DA ratio and TH activity increased again to reach the diestrus levels. In DMBA-pretreated females, similar fluctuations in TIDA neuronal activity occurred during the estrous cycle, but the dynamics of these changes was altered: the DOPAC/DA ratio and TH activity first showed a marked increase in the morning of proestrus day, before decreasing dramatically.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in interleukin-1beta mRNA expression in the rat ovary during the estrous cycle in response to lipopolysaccharide.

The changes in interleukin-1 (IL-1) beta mRNA expression and the number of macrophages were studied in the ovary during the estrous cycle in rats and after intraperitoneal injection of lipopolysaccharide (LPS, 2 mg/body) 2 hours before autopsy. IL-1beta mRNA expression was very low in the ovary, and there was no statistically significant change during the estrous cycle. Hybridization signals of IL-1beta mRNA were localized intensely in the thecal layer, moderately in the corpora lutea, and slightly in granulosa cells of the ovary during the cycle. The number of macrophages seen mainly in the hilum and interstitium significantly increased on proestrus compared with other estrous days. LPS significantly increased IL-1beta mRNA expression on each day with the highest response to LPS at 1500 h on proestrus, and caused an increase in the number of macrophages in the ovary within 2 hours. These results indicate that IL-1beta mRNA expressions are low during the estrous cycle in rats, and proestrus is the day of maximal IL-1beta synthesis in response to LPS. The increase in IL-1beta synthesis caused by LPS might be due to at least the influx of macrophages into the ovary.     

Variations in the hypothalamic-pituitary-adrenal response to stress during the estrous cycle in the rat.

To investigate the role of gonadal steroids in the hypothalamic-pituitary-adrenal (HPA) response to stress, we studied adrenocorticotrophin (ACTH) and corticosterone (B) responses to 20-min restraint stress in cycling female rats, and in ovariectomized (OVX) rats replaced with physiological levels of estradiol (E2) and progesterone (P). In cycling rats, we found significantly higher peak ACTH (P less than 0.01) and B (P less than 0.05) responses to stress during proestrus compared to the estrous and diestrous phases. No differences were found in either basal ACTH and B levels across the cycle phases. In a separate study, OVX rats were maintained on low, physiological levels of E2 and P with silastic implants for 3 days, and injected either with oil (O'), 10 micrograms of E2 (E') 24 h before stress testing, or with E2 and 500 micrograms P 24 and 4 h, respectively, prior to stress (EP'). These treatments mimicked endogenous profiles of E2 and P occurring during diestrous, proestrous, and late proestrous-early estrous phases, respectively. In response to stress, ACTH levels were higher (P less than 0.01) in the E' group compared to the EP' and O' groups. Although the peak B response was similar in all groups, the E' and EP' groups secreted more B after the termination of stress than did the O' group. Within the 20 min stress period, ACTH levels in the E' group were significantly (P less than 0.05) higher at 5, 10, and 15 min after the onset of stress, compared to the EP' and O' groups. Plasma B levels were significantly higher in the E' group at 5 and 10 min (P less than 0.05 and P less than 0.01, respectively) compared to the EP' and O' group. beta-endorphin-like immunoreactive responses to restraint stress were also significantly higher in the E' group compared to the EP' (P less than 0.05) and O' (P less than 0.01) groups. In contrast to the effect seen at 24 h, ACTH responses to stress 48 h after E2 injection in the E' group were comparable to O' animals. There was no effect of E2 on ACTH clearance, whereas B clearance was enhanced in E' treated animals vs. O'-treated animals. These results indicate that the HPA axis in the female rat is most sensitive to stress during proestrous. Such enhanced HPA responses to stress are limited to the early portion of proestrous, as progesterone appears to inhibit the facilitatory effects of estrogen on ACTH release during stress. Taken together, these results suggest an ovarian influence on both activational and inhibitory components of HPA activity.     

Changes in the pulsatile pattern of luteinizing hormone secretion during the rat estrous cycle

To ascertain whether changes in the pattern of GnRH release from the hypothalmus occur during the 4-day rat estrous cycle, the pattern of LH release was characterized on each day of the estrous cycle, and the results were interpreted in light of the changes in pituitary responsiveness to GnRH previously described by this laboratory to occur during this time. Blood samples were taken from intact, freely moving rats via venous catheters at 6- to 10-min intervals for 3-4 h. LH pulse height and LH interpulse interval were quantified on each day of the cycle, and the transition on the afternoon of proestrus from tonic LH release to the preovulatory LH surge was detailed. The effects on the pattern of LH release during estrus of small doses of GnRH (0.4 ng) and the continuous infusion of the opioid antagonist naloxone were also examined. Plasma LH concentrations (NIAMDD rat LH-RP-1) were determined with a highly sensitive LH RIA. LH pulses were identified using the PULSAR algorithim. The LH interpulse intervals of 46 +/- 2 min on diestrous-1 day, 49 +/- 4 min on diestrous day 2, and 60 +/- 8 min on proestrus immediately before the LH surge were not significantly different. There were no changes immediately preceding the preovulatory LH surge on the afternoon of proestrus in either the LH interpulse interval or the LH pulse height. Instead, the transition from tonic LH secretion to the preovulatory LH surge was found to occur abruptly. These data suggest that an abrupt increase in GnRH secretion during the afternoon of proestrus initiates the dramatic rise in LH concentrations. The pattern of LH secretion during the day of estrus differed significantly from that on the other days of the cycle in that no LH pulses were observed. However, the administration of small pulses of GnRH elicited physiological elevations in LH release. Furthermore, the continuous infusion of naloxone to estrous rats immediately stimulated a pulsatile pattern of LH secretion, with a LH interpulse of 56 +/- 4 min. These data indicate that the absence of LH pulses during estrus may result from a deficit in GnRH release. Similar modifications in GnRH release during the other days of the cycle were inferred from the observed changes in LH pulse heights. The LH pulse height of 21 +/- 3 ng/ml on diestrous day 2 was significantly less than the LH pulse height of 41 +/- 4 ng/ml on diestrous day 1 or 35 +/- 4 ng/ml on proestrus before the surge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anterior pituitary dopamine receptors during the rat estrous cycle. A detailed analysis of proestrus changes

A method for the enzyme immunoassay (EIA) of delta sleep-inducing peptide (DSIP) has been developed and applied to determine the metabolic clearance rate and biological half-life of DSIP administered to dogs. Antisera to DSIP conjugated to bovine serum albumin were raised in rabbits and proved to be specific for the C-terminus of the peptide. DSIP conjugated to horseradish peroxidase served as the labeled antigen in the EIA and enzyme activity was determined by fluorophotometry. The assay sensitivity was approximately 30 pg/ml. 1 or 2 mg of DSIP was injected intravenously into 4 anesthetized dogs and blood was taken at 5-min intervals. Unextracted plasma was subjected to the EIA directly and showed parallel displacement curves to the standards. DSIP was found to have a rapid disappearance with a mean metabolic clearance rate of 30.7 +/- 2.5 ml/kg . min and a mean half-life of 4.0 +/- 0.7 min in the dogs. Additional measurement of the metabolic parameters in a monkey and 3 rats treated similarly revealed a rapid in vivo clearance of DSIP from plasma with a half-life of 2.9 and 2.0 +/- 0.54 min, respectively.     

Changes in the number of GnRH-receptive cells during the rat estrous cycle: biphasic effects of estradiol

The number of gonadotropin-releasing hormone (GnRH) receptors is known to vary throughout the estrous cycle and in other endocrine states in the rat. These changes in receptors parallel closely the concentrations of serum estradiol during the cycle. In the present study, we used two different cytochemical techniques to determine if changes in GnRH receptors represented alterations in the number of GnRH-receptive cells. Furthermore, we tested the effects of estradiol pretreatment on this phenomenon. Dispersed pituitary monolayers taken at different stages of the cycle were stimulated for 3 min with 1 nM biotinylated [D-lys6]-GnRH (bio-GnRH) which was localized using the avidin-biotin-peroxidase complex (ABC) technique and a black peroxidase substrate. Parallel groups were stained, while living, with an avidin-fluorescein conjugate. Some monolayers were pretreated with physiological concentrations of estradiol benzoate (1 nM-1 pM) prior to bio-GnRH exposure and ABC stains. The resulting stains demonstrated that the percentage of bio-GnRH-receptive cells was 2-3 times greater at 10.00 h proestrus (20.2 +/- 4%) when compared to the same time in estrus (8.7 +/- 2%), diestrus I (5.2 +/- 0.5%), diestrus II (7.6 +/- 1%), and at 17.00 h diestrus II (11.4 +/- 0.9%) and proestrus (7.4 +/- 0.8%). These data correlated well with those obtained from living gonadotropes stained with avidin-fluorescein. Estradiol exerted a biphasic effect dependent upon the stage of the cycle at which the cells were taken.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the prolactin response to thyrotropin-releasing hormone (TRH) during the menstrual cycle of normal women.

Serum prolactin (PRL) and thyrotropin (TSH) levels were measured after iv administration of 200 microng of synthetic thyrotropin-releasing hormone (TRH) in 20 normal women ages 18 to 34. Ten women received TRH on days 7 to 8 of the menstrual cycle and 10 women received TRH on days 21-22. Although there was no difference in the dose of TRH relative to body weight in the two groups of women, the peak PRL level after TRH stimulation was greater in the women studied on day 21-22 (48.5+/-5.7 ng/ml, mean+/-SE) than on day 7-8 (35.2+/-4.2 ng/ml) of the cycle (P less than 0.05). In contrast, TSH rose to a greater degree in the preovulatory phase (13.8+/-1.8 micronU/ml) than the luteal phase (7.7+/-0.7 micronU/ml of the cycle (P less than .01). Studies of the PRL and TSH response after TRH administration should take the phase of the menstrual cycle into account.     

Variations of liver prolactin receptors during the estrous cycle in normal rats and in the genetically hypoprolactinemic IPL nude rat

Ovine prolactin (oPRL) binding to liver membranes was studied during the estrous cycle in normal and in genetically hypoprolactinemic rats. Serum levels of hormones were measured by radioimmunoassay and prolactin (PRL) binding was determined using 125I-ovine PRL in the 100,000 X g pellet. Scatchard plots obtained were curvilinear throughout the estrous cycle in the normal rat. They were analyzed in reference to the co-operativity model and to the Hill model which give the factor delta and Hill's coefficient (nH), respectively. During the estrous cycle, delta values varied from 3.77 +/- 0.66 on the day of estrous to 13.48 +/- 1.34 on the day of proestrus at 16.00 h. At the same time, nH were 0.97 +/- 0.033 on the day of estrus and 0.72 +/- 0.025 on the day of proestrus at 16.00 h. On the other hand, the number of PRL receptors did not change significantly throughout the estrous cycle. Moreover, the dissociation of 125I-oPRL from its receptor was accelerated by the presence of native ovine oPRL. These results suggest the presence of a negative co-operativity which reached a maximum on the day of proestrus in the normal rat. This co-operativity during the estrous cycle was not found in liver from genetically hypoprolactinemic (IPL nude) rats, which present a total absence of lactation. The delta values did not vary significantly and were 6.52 +/- 1.30 on the day of estrus and 4.41 +/- 0.52 on the day of proestrus at 16.00 h. The difference between the two rat strains was statistically significant on the day of proestrus at 16.00 h for both delta and nH values.(ABSTRACT TRUNCATED AT 250 WORDS)